Waters XBridge Columns - Manual (Page 2)

[ Care and Use ManUal ]
XBridge ™ Columns 2
I. Get tInG started
Each XBridge ™ column comes with a Certificate of Analysis and a Perfor-
mance Test Chromatogram. The Certificate of Analysis, located on the technical
information CD, is specific to each batch of packing material contained in
the XBridge
™ column and includes the batch number, analysis of unbonded
particles, analysis of bonded particles, and chromatographic results
and conditions. The Performance Test Chromatogram is specific to each
individual column and contains the information: batch number, column
serial number, USP plate count, USP tailing factor, retention factor, and
chromatographic conditions. This data data should be stored for future
reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 5 µm pack-
ing in a 4.6 mm i.d. column. Scale the flow rate up or down accordingly based
upon the column i.d., length, particle size and backpressure of the XBridge
™
column being installed. See Scaling Up/Down Isocratic Separations section
for calculating flow rates when changing column i.d and/or length. See
Connecting the Column to the HPLC for a more detailed discussion on HPLC
connections
1. Purge the pumping system of any buffer-containing mobile phases and
connect the inlet end of the column to the injector outlet. An arrow on
the column identification label indicates the correct direction of solvent
flow.
2. Flush column with 100% organic mobile phase (methanol or ace-
tonitrile) by setting the pump flow rate to 0.1 mL/min. and increase
the flow rate to 1 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column outlet, stop
the flow and attach the column outlet to the detector. This prevents
entry of air into the detection system and gives more rapid baseline
equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved, pro-
ceed to the next section.
Note: If mobile phase additives are present in low concentrations (e.g.,
ion-pairing reagents), 100 to 200 column volumes may be required for
complete equilibration. In addition, mobile phases that contain formate
(e.g., ammonium formate, formic acid, etc.) may also require longer initial
column equilibration times.
b. Column Equilibration
XBridge ™ columns are shipped in 100% acetonitrile. It is important to ensure
mobile phase compatibility before changing to a different mobile phase sys-
tem. Equilibrate the column with a minimum of 10 column volumes of the
mobile phase to be used (refer to Table 1 for a listing of empty column
volumes).
To avoid precipitating out mobile phase buffers on your column or in your
system, flush the column with five column volumes of a water/organic
solvent mixture, using the same or lower solvent content as in the desired
buffered mobile phase. (For example, flush the column and HPLC system
with 60% methanol in water prior to introducing 60% methanol/40%
buffer mobile phase).
For XBridge HILIC columns, flush with 50 column volumes of 50:50
acetonitrile:water with 10 mM final buffer concentration. For XBridge
HILIC Amide columns, flush with 50 column volumes of 60:40
acetonitrile:aqueous. Prior to the first injection, equilibrate with 20 col-
umn volumes of initial mobile phase conditions (refer to Table 1 for a list
of column volumes). See “Getting Started with XBridge HILIC Columns”
or “Getting Started with XBridge HILIC Amide Columns” for additional
information.
c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it in the desired
application. Waters recommends using a suitable solute mixture, as found
in the “Performance Test Chromatogram,” to analyze the column upon
receipt.
2. Determine the number of theoretical plates (N) and use this value for periodic
comparisons.
3. Repeat the test at predetermined intervals to track column perfor-
mance over time. Slight variations may be obtained on two differ-
ent HPLC systems due to the quality of the connections, operating
environment, system electronics, reagent quality, column condition
and operator technique.